Neural stem cells have been isolated from various regions of both rodent and human nervous systems and expanded in vitro as free floating aggregates (U.S. Pat. No. 5,851,832 to Weiss et al.) or as a monolayer attached to a substratum-coated dish (U.S. Pat. No. 5,753,506 to Johe). Neural stem cells are capable of extended self-renewal and have the ability, under appropriate conditions, to generate one or more subtypes of neurons and glia cells in vitro. By virtue of these properties, neural stem cells and their progeny can be applied for pharmaceutical drug discovery and for cell transplantation in neurological disorders.
The fate of neural stem cells can be controlled by a variety of extracellular factors. Growth factors such as basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (U.S. Pat. Nos. 5,851,832 and 5,753,506) and Leukemia Inhibitory Growth Factor (LIF; U.S. Pat. No. 6,103,530 to Carpenter) have all been shown to control the in vitro proliferation of neural stem cells. Growth factor expanded stem cells can differentiate into neurons and glia after mitogen withdrawn from the culture medium, but the proportion of neural stem cells that differentiate into neurons is minimal. However, the proportion of neural stem cells differentiating into the neuronal pathway can be increased by exposing the neural stem cells to various other growth factors such as Platelet Derived Growth Factor (PDGF; U.S. Pat. No. 5,753,506); bFGF (U.S. Pat. No. 5,766,948 to Gage and Jasodha); Brain Derived Growth Factor (BDNF; Shetty and Turner, J Neurobiol. 1998;35:395–425); including neurotrophins such as Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4; Caldwell et al., Nat Biotechnol. 2001;19:475–9); Notch antagonists (U.S. Pat. No. 6,149,902 to Artavanis-Tsakonas et al.), retinoic acid (U.S. Pat. No. 6,395,546 to Zobel et al.; or BMP-2 (U.S. Pat. Nos. 5,948,428 to Lee et al., and 6,001,654 to Anderson et al.) to increase the quantity of neurons derived from a certain amount of neural stem cells.
The antiepileptic drug valproate (VPA) or valproic acid (n-dipropylacetic acid), a simple branched-chain carboxylic acid, is an established human teratogen, (i.e., an agent capable of causing malformations in embryos), which affects neural development in human fetuses exposed to the drug during early pregnancy. In vitro cellular models have been used to investigate the teratogenic effect of valproate and its structural analogs, mainly by use of tumor cell lines (Courage-Maguire et al., Int J Dev Neurosci 1997; 15:37–43). In such studies, valproate has been found to inhibit the proliferation and block retinoic acid-induced neuronal differentiation of the teratocarcinoma cell line NTera-2 (Skladchikova et al., Neurotoxicol. 1998;19:357–70). By contrast, in a neuroblastoma cell line, the antiproliferative effect of valproate appeared to be associated with cell differentiation, as an increase of neuronal morphology and neural cell adhesion molecule expression was observed after exposure to valproate (Cinatl et al., Anticancer Drugs 1996;7:766–73; Slesinger and Singer, Epilepsia 1987; 28:214–21; Knupfer et al., Anticancer Res 1998; 21:347–51; Yuan et al, J Biol Chem 2001; 276:31674–83). The reported effect of valproate on maturation in these studies was always associated with an inhibition of cell proliferation. Similar effects of valproate have been found in other tumors (Slesinger and Singer, Epilepsia 1987; 28:214–21; Knupfer et al., Anticancer Res 1998; Gottlicher et al., Embo J 1998; 20:6969–78). In a recent study, valproate was also shown to increase the cytoprotective protein bcl-2 in the brain and neurite outgrowth in vitro (Manji et al., Biol Psychiatry 2000;48:740–54), indicating that valproate may have a neurotrophic effect on already differentiated neuronal cells, i.e., to inhibit apoptosis and promote maturation of neurons. Finally, valproate has been suggested to serve as a neurotrophic factor and an anti-proliferative compound on tumorigenic and primary neuronal cell lines (U.S. Pat. No. 5,672,746 to Nau et al.). There have been no reported results showing that valproate induces neuronal differentiation without inhibiting proliferation.
The availability of large quantities of neuronal cells such as neurons or neuroblasts is important for the application of such cells both in vivo and in vitro, e.g., in cell transplantation therapy for neurodegenerative diseases and in vitro drug testing in psychiatric disorders. Such quantities can only be obtained by differentiation of cultured neural stem cells into these cell types, which requires large-scale cell culture using a significant amount of various growth factors and/or neurotrophins. Given the sparse availability of neural stem cells, especially human neural stem cells, and the cost of growth factor and neurotrophin preparations, there is a need for more efficient and economically viable strategies for differentiating neural stem cells into neuronal cells such as neuroblasts and neurons. This invention addresses these and other needs in the art.